Hyaluronic Acid Production: Market Analysis, Strain Selection, Fermentation Process Setup And Optimization

Market Analysis of Hyaluronic Acid

The hyaluronic acid (HA) generally remains stored in the body in inactive form. The production of hyaluronic acid is used to make various cosmetic products, including moisturizer and hair serum. Chemical property of hyaluronic acid is to bind with the water molecule to enhance the water content in the skin. It creates a barrier to slow down the evaporation process through hydration and receives the water content from environmental humidity. The wide applications of HA are implicated in biomedical, food, health, and cosmetic fields. Formally hyaluronic acid was extracted by Karl Meyer and John Palmer in year 1934 that was isolated from tissues of bovine vitreous humor. However, the production in recent years is made through wide varieties of microbes; such as species of streptococcus and lactobacillus bacteria (Murado et al. 2012). The microbes are carried to the fermenter where they are amplified by providing the favourable growth conditions and the nutrients so that bacteria can use the substrate to release the products (HA). Further discussion will highlight on the market analysis, strain selection, fermentation process setup, fermentation scale-up, and fermentation process optimisation.

Market analysis of hyaluronic acid is based on the wide application of hyaluronic acid in the market. Traditional formation of HA occurred through rooster comb-like animal tissues. Although, the tissues of all the living species tend to generate HA, the yield from tissues is inhibited because of the production of hyaluronidase enzyme that perturbs the human immune system and hinders the high HA production through hydrolysis process (Murado et al. 2012). However, the recent biological production of hyaluronic acid comprises microbial fermentation (Flores-Méndez et al. 2021). Microbial HA production is highly Effective to cost, produce less environmental pollution than the former HA production process and uses the easy handling of biological process on the industrial scale (Rossatto et al. 2022).

• Market potential of hyaluronic acid

A report from the American Society of Aesthetic Plastic Surgery (2004) provides data on using HA in various clinical products for the patients’ well-being. Approximately 23 000 dermatologists, plastic surgeons, and cosmetic surgeons annually perform 11.8 million surgeries annually, generating $12.5 billion in fees. However, by 2011, the dermal filler market has expanded at an annual rate of about 25%, reaching $1.5 billion in global sales. A new era has ushered the dermal enhancement by launching QMed’s Restylane along with NASHA technology. NASHA technology stands for Non-Animal Stabilized Hyaluronic Acid, producing filler by utilizing the microbial load to produce HA. NASHA technology also addresses shelf life, animal origin, and skin testing (De Maio and Rzany 2014).

The increased applications of HA have made the market growth over the years, with a current spending value of USD 1000–5000/kg of HA products. A firm Research of Transparency Market, 2014 provided data on the marketing value of the HA, which has reached USD 9.05 billion by 2019. Moreover, the expected range till 2027 is going to reach USD 16.6 billion.

• The competitors on the market

Hyaluronic acid is a body moisturized serum that draws water from the deeper dermis, carries it to the epidermis, and increases skin elasticity (Qiu et al. 2021). There is subsequent internalization of the competition among companies to produce hyaluronic acid, including pharmaceutical companies, nutraceutical industries, and cosmetic industries. The value of HA production has reaches to 1.0 billion dollars in year 2020, which is targeted for further projecting to reach 2.1 billion dollars by year 2030. The Research and Markets is the largest market research companies of the HA manufacturing that includes many countries including China, United States, UK, Italy, Japan, India, and Brazil.

• The target market using HA

Applications of Hyaluronic Acid

The successful production of HA on an industrial scale with Streptococcus sp. summarizes the research history and current commercial market. Streptococcus zooepidemicus is the natural HA producer ranging the current worldwide market for HA over $1 billion. List of some medical applications of HA in patients journey includes osteoarthritis treatment, plastic surgeries, dermatology (including anesthetic and drug delivery), urology, cancer studies, otolaryngology, and rheumatology (Huynh & Priefer, 2020). Since 2000, 15 million of the knee osteoarthritis patient population required viscosupplements through HA uses with increased HA demand up to viscosupplements. The first viscosupplementation product was injected into the osteoarthritis patient in US, which then gained rapid acceptance by physicians because of its convenience (Liu et al. 2011). viscosupplementation market of HA is being involved in providing a higher and more effective treatment regimen through European industrial productions and has undergone the convenient procedure to attract more patients. The Asian patients, who are affected by the aging and physically active demographics, have also shifted towards using HA viscosupplementation, which is rising treatment’s awareness and benefits among physicians.

Another major use of HA is identified in the plastic surgery field, with an increased global market for dermal fillers. According to medical Insight Inc, the dermal fillers market is ground to be approximately 759 million USD in 2009, with a range of 100 different dermal fillers. More than half of such blooming global filters are based on HA productions (Kang et al. 2017).

The candidate microbes and reason of choosing these microbes

Two candidate microbes selected for the production of Streptococcus zooepidemicus and Lactobacillus acidophilus.

• The strain selection is based on the availability of the following factors:
  • Availability of the strains: the microbes are utilized based on GRAS (generally recognized as safe) status, which means that the chosen microbe is not toxic/pathogenic and has the ability to produce a high-quality HA product with a great market value. Quality of HA can be determined by its market values and low cost in purification process, making HA production higher and faster. HA production on an industrial scale utilizing microbes was firstly performed by Shiseido in the year 1980, which uses zooepidemicusfor HA production first time. For the elevated HA production from other natural producer, streptococci, the many other bacteria are also being utilized through genetically engineered processes.  
  • The molecular weight of products: The molecular weight of hyaluronic acid comprises 6 million Daltons. It possesses the characteristics of lubricity and hydrophilicity, high viscoelasticity, biocompatibility, and high moisture retention capacity. The reason for HA formulation through streptococcal fermentation is that it produces a high amount of HA through its metabolic process. Among the formation of HA and microbial cell growth studies, there exists a strong competition as both the sources share a common precursor, including UDP-glucuronic acid and UDP-N-acetyl-glucosamine (Qiu et al. 2021).
  • Yield: hyaluronic acid (HA) is generally a type of rigid biopolymer belonging to the clads ofpolysaccharide, which is linear polymer and unbranched high molecular weight. The GRAS microbes highly mediate yield of HA. Generally, to produce a molecule of HA dimer, the microbe should release at least two NAD⁺, two UTP, three ATP molecules, one Acetyl-CoA, and one glutamine molecule through the glycolytic pathway. Hence, the best alternative used within Streptococci species, Lactobacillus acidophilus, is investigated as GRAS microbe to produce HA (Chahuki et al., 2019).

The production range of HA by S. zooepidemicus reaches 6 to 7 g/L HA, if favourable climatic conditions and suitable media preparation methods are being implicated in the culture conditions. The selected strain for Lactobacillus acidophilus is PTCC1643, which can produce approximately ~0.25 g per litre of HA, even without any genetic modification. Further medium optimization through response surface methodology (RSM) by co-expressing (HA synthase or has) hasA and hasC genes, a fourfold increase of approximately 1.7 g L−1 can be done. Thus, genetic modifications and medium optimization are necessary for the production of increased HA production (Manfrão?Netto et al. 2022).

• Cost, medium, fermentation period, temperature, downstream processing:To commercial production of HA is being produced through microbial fermentation to reduce the cost of high purification process and harsh extraction conditions needed for the extraction from animal tissues. For a selected strain of zooepidemicus (ATCC 35246), the culture medium included the inoculation Glucose 60 g per liter and a chemically defined medium or CDM. However, the fermentation mode will occur through batch carrying a capacity of 2 L and 600 rpm of aeration parameters. Maintaining all the parameters with subsequent handling procedures produces a production of 4.2 g/L HA. The molecular weight of 4.2 g/L HA will be 3.2 × 106 Da (Liu et al. 2011). Downstream processing is the essential step of fermentation, which is defined as maintaining the fermentation process, which requires various intrinsic and extrinsic factors to be optimized.
• Possibilities to be further optimized:Whether recombinant strains of Lactobacillus acidophilus or zooepidemicus are effective strains to produce HA, they have some limiting factors affecting HA production, such as cost of raw materials and production cost. Hence, further optimization is necessary to improve the HA yield and molecular weight. Replacements are needed in the manufacturing process to reduce cost and enhance productivity. The cost of the product formation can be reduced by more than 30% by using cheap crude materials or conversion of renewable/ agricultural resources. Other methods include the metabolic flux and control analysis throughout biochemical reactions to enhance HA concentration.

• Weakness of the candidate microbes and potential plans to improve it

The formation of HA and cell growth of the microbes both rely on the same precursors in the fermenting medium, which are UDP-glucose, UDP-N-acetylglucosamine, and glucose-1-phosphate. Hence, favorable HA production may not form. Moreover, the biomass formation process gets reduced due to the competition of carbon flux for cell growth and HA synthesis. By weakening the glycolytic pathway and biomass rate reduction can tend to increase the HA molecular weight as well as HA titer. The incorporated intermittent alkaline stress strategy that includes the rise in pH from 7 to 8.5 for half a day can help in enhancing the biomass formation up to 6.5 g/L.

Moreover, some of the Streptococcus sp. are also do not stand on the GRAS status, which is why industrial companies are facing a growing concern due to pathogenic species of streptococci. Hence, before utilizing these microbes, additional costing is required to purify these strains.

• The top 1 candidate used in the following design:Lactobacillus acidophilus is a GRAS microbe that produces a high amount of HA. Hence, Lactobacillus acidophilus can be effectively utilised in HA production. However, the maximised yield can be produced from streptococcus species fermentation. Although, some strains of zooepidemicus can release toxic by-product in the fermenter affecting the yield, still it is highly preferred and recommended bacteria among two microbes, S. zooepidemicus, and Lactobacillus acidophilus. Streptococcal fermentation is recommended due to the high moisture retention, excellent viscoelasticity, wide range of applications in the fermentation process, and easy handling. Preparation of the fermentation process is maintained by the media composition and culture inoculum. Moreover, the genetic modification of the bacteria has made it the ideal microbe for HA manufacturing. Genetic modifications occur in the hyaluronic acid synthase enzymes to increase productivity (Manfrão?Netto et al. 2022).

Microbial Strain Selection for HA Production

The process set up in the fermenter is maintained to initiate the HA production by incorporating the bacteria and including all the necessary parameters for the bacteria growth. Hence, the Process of HA production through the bacterial metabolic process is described below:

The sugar backbone of HA is a derivative of glucose-6- phosphate and fructose-6-phosphate that undergoes two sets of reactions to formulate HA. Glucose converts to glucose 6 phosphate in the presence of phosphoglucomutase enzyme, which further converts into UDP-glucose. The enzyme utilized in UDP-glucosepyrophosphorylase (or hasC). UDP-glucose converts into UDP-glucose-6-dehydrogenase (or hasB) enzyme in the presence of other enzymes called UDP-gluconic acid. The final product from UDP-gluconic acid to hyaluronic acid occurs in the presence of hyluronase synthase (or hasA) enzyme releasing the UDP precursor. However, the second reaction is the conversion of glucose-6-phosphate that converts into fructose-6-phosphate in the presence of enzyme phosphoglucoisomerase or hasE. fructose-6-phosphate then converts into glucosamine-6-P (utilizing enzyme aminotransferase). The produced substrate, glucosamine-6-P, then transforms into glucosamine-1-P in the presence of phosphateglucosamione mutase. Further conversion of glucosamine-1-P occurs to form N-acetylglucoseamine-1-P product in the presence of enzyme acetyltransferase (has). N-acetylglucoseamine-1-P transforms into UDP-N- acetylglucosamine by using enzymes UDP-N- acetylglucosamine phosphophosphorylase (has) enzyme. UDP-N- acetylglucosamine is the constituent polymer of the bacterial cell wall and acts as the second HA precursor (de Oliveira et al. 2016).

 https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0517-4 

Fig. 1: the given flow chart provides a good understanding of the reaction process of HA synthesis from bacteria metabolic process undergoing glycolytic pathway

• Optimal temperature: Optimal temperature for the growth of bacteria is 37 degrees Celsius, which is carried for 16 hours so that enough of the cell growth can take place on the media to take the microbial load for the fermentation process.
• Medium composition: A basal medium composition is required to grow the bacteria which will be incorporated into 100 mL of the media composition process, including the inoculum of the following ingredients:

A single type of colony grown on the brain heart infusion agar at room temperature for approximately 16 hours. Now, the cultivation of the fermenter carries a working volume of 1.4 liter contained in the 2L glass vessel. The other parameters maintained in fermenter include the measurement of pH, controlled temperature and dissolved oxygen. Further process of fermentation is agitation that occurs at 300 to 400 rpm (rotation per minutes). The maintained pH is 7.0, which can be automated by adding 5 M NaOH. After 10 hours of the fermentation process, 60 percent of glucose is also added to maintain the media composition. The reason of adding more glucose is that enough of the glucose might be utilized by the microbes present in the fermenter. The concentration of glucose in the media is determined through a glucose assay kit (Sigma). After 24 hours, a measurement of the bacterial presence can be performed by taking the optical density of the culture at 600 nm using spectrophotometer (Wakade et al. 2017).

• Fermentation type: The fed-batch type of fermenter is the most suitable bioreactor for the production of HA through the provided microbial load, zooepidemicusspecies. The main reason of using a fed batch fermenter is that it possesses sufficient bioactive metabolites, limited oxygen transfer through inlets, and controlled microbial growth (Shahira 2019). In a fed-batch type of fermenter, the fresh feeding is most appreciated, as, after a sufficient interval of time, the bacterial metabolic process results in enough glucose consumption. Thus, the glucose concentration or other parameter get affected in the fermenter, reducing the capacity of HA yield. However, by utilizing the fed-batch fermenter, parameters can be maintained by immediate changes in intrinsic or extrinsic factors. Moreover, it has an extended culture lifespan and significant cell concentration.

Extraction of HA from bacteria has been a steadily increasing process over the last two decades. The desired bacteria are grown in the fermenters to produce the natural HA. Currently, lactobacillus and streptococcus species are highly used bacteria in producing HA. The enzyme complex of streptococcus is crucial in the formation of HA.

Potential concerns of HA

• The production of HA is based on the extraction of HA from large-scale bacterial fermentation. The commercial interests to produce rapid and safer HA, non-pathogenic microbes are chosen. Although, the reason for choosing streptococcus is the production a high source of HA. Streptococcus is the pathogenic microbe that secretes toxins in fermenter.
• A competition of Streptococcus zooepidemicus occurs in the fermenter along with the biomass production. Streptococcus zooepidemicus tend to compete for the utilization of enough of the carbon source in order to form the biomass production and lactate formation.
• The hasenzymes are the integral membrane proteins, which are lipid dependent. Hence, the extractions of the enzymes are quite difficult (Sze et al. 2016).
• For the production of high yield of HA and lactic acid, an inverse relationship can also be seen, as both of them are dependent on the glucose utilization.
• Elimination of encountering issues to increase the bio productivity of HA
  • The toxin creating genes or proteins must be eliminated to make the safer alternative generating a larger HA amount. The production generally enhanced through the culturing of genetically modified bacteria. The modification can be performed through the co-expression of uridine diphosphate–glucose dehydrogenase or UDP-GlcDH and HA synthase enzyme (Liu et al. 2019).genetically modified bacteria can produce HA more than 1 MDa. Moreover, the benefits of using GM microbes can increase the half-life of the molecules by maintaining the physiological properties of the molecule (Liu et al. 2011).
  • Redirecting the carbon flux in the fermenter can enhance the molecular weight of HA in fermenter. However, in order to redirect the carbon flux, glycolytic pathway is initially inhibited(Shah et al. 2013). Moreover, molecular weight of HA up to 0 MDa can also be increased by the addition of antioxidant tannic acid. Addition of some amino acids, including glutamine (amino acid) and iodoacetate (a derivative of acetic acid), is very effective that can increase HA production by increasing its molecular weight up to 150%.
  • The activity of toxin releasing hasoperon genes that can be downregulated to overcome the lipid-dependent drawback of enzymes through the isolation and purification processes. The active detergents are incorporated in the process to solubilize the has membrane-bound proteins. However, the solubilized deterges should not contain the active compounds that can impair the HA productivity or functions. The purified enzymes retain the ability to synthesize the natural form of HA. Moreover, in order to produce a high molecular weighted HA, a cell-free system is being utilized that must contain a low polydispersity and controlled processivity (Prasadet al. 2012).
  • In order to overcome the shortage or competition in HA and lactic acid production by using glucose, more of the glucose sources are being added to the fed-batch fermenter within 12 to 16 hours, where most of the glucose is being supposed to be utilized by the microbes.

https://www.sciencedirect.com/science/article/abs/pii/S1369703X13002544 

Fig. 2: The improved yield of HA achieved by incorporation of glutamine amino acid.

The factors which are required to be optimizedinclude aeration of Streptococcus zooepidemicus ATCC cultures, nutrient sources, temperature, agitation, and the purification stages. The adjustment in such parameters to the optimal range can provide structural modification in the bacterial cells, which ultimately can increase the molecular weight of HA (Rossatto et al. 2022).

Optimization for hasA enzyme: hasA enzyme is the crucial enzyme for the production of natural HA by the incorporation of microbes in the medium. Streptococcus zooepidemicus is the main bacteria containing operon encoding the hasA enzyme used for HA synthesis. HasA adds two HA precursors, including UDP-GlcNAc and UDP-GlcUA that directly include in the glucuronic acid and N-acetylglucosamine production for the growing HA chain (Oliveira et al. 2016). The optimization of the enzyme can be mediated by the provision of high ATP concentration. The higher production of ATP can occur through the incorporation of glucose (Weigel, P.H., 2015).

Fermentation Process Setup and Optimization

Optimization for pH conditions: The optimum pH to increase the production rate and yield of the hyaluronic acid can be done by providing the optimal pH 7.7±0.2 under the aerobic condition. At the lower pH, below 6 or more than 8 pH, can affect productivity due to alteration in bacterial growth (Cavalcanti et al. 2020).

Optimization for aerobic conditions: Oxygen is the key factor for bacteria to grow and reproduce so that a higher production of HA can take place in the fermenter. Although, an insufficient level of oxygen can reduce bacterial accumulation. Hence, during the process of HA production in fermenter, aerobic conditions are effectively optimized.

Optimization for agitation speed: agitation speed is the next key factor affecting the yield of HA generation from bacterial extraction. Although agitation is not directly related to the HA production process, it makes the bacteria available for the extraction of HA during the fermentation process.

• Design experiments: Designing the experimental process is performed for sufficient HA productions and monitoring the fermentation conditions. The process is maintained through the incorporation of several variables, which tend to increase HA production. Following procedures are required to design the experiment
  • Culture maintenance for the production of approximately 1 liter of HA: The prepared culture can be maintained for a longer time by storing it at -80oC. At the time of fermentation, the inoculum of culture is transferred into the fermenter. The concentration of thesugars is made according to the volume of the medium in which cell cultures will be maintained (Aroskar et al. 2012). The normal glucose concentration for the bacteria to grow effectively is 30 g L-1. The addition of sugar is made within 12 hours of glucose incorporation, as it gets consumed by bacteria. Similar to glucose concertation, yeast extract is also utilized by the bacteria, which is needed to be maintained at 30 g L-1. The optimal pH range is set at 0, which can be increased or decreased by 1 (ranging 6, 7, and 8). However, the best activity of bacteria can be seen on 8.0 pH. The temperature range of the fermenter is maintained at 37^oC, which even changes from 34 to 40, which will be considered the optimal range. Nutritional parameters are the standard parameters affecting the overall productivity. The incorporation of nutritional parameters including Lactate, formate, and acetate is also essential for the enough HA production. The provided agitation speed for 30 g L-1 glucose media is approximately 200 rpm (Pan et al. 2015).

Summary: The first and original HA development was supposed to be used in clinical medicine as a first non-inflammatory, highly purified form extracted from the umbilical cords and rooster combs. However., the current focus of HA production is through microbial fermentation. In microbial fermentation, microbes are grown in the laboratory on a large scale, as it is very beneficial than the bovine vitreous humor fermentation. Hence, microbial production is highly recommended and is under legislations. The current and increased applications of HA have reached a current value of USD 1000–5000 USD per kg of HA. The major uses of HA productions on the market analysis are needed on the clinical basis due to the production of high viscosity from HA. viscosupplementation market of HA is being involved in providing a higher and more effective treatment regimen through European industrial productions and has undergone the convenience procedure to attract more patients. HA producing bacteria in culture media must be ensured that chosen bacteria (basically streptococcus and lactobacillus bacteria) maintain the properties of GRAS status. Several alternatives, methods are available to increase the productivity of HA that can be applied in various companies. However, the main focus is being paid to the parameters affecting the cell growth of the microbes. Subsequent parameters are being adjusted for the focused increased optimization of cultivation conditions and culture media, including Fed-Batch Fermentation, aerobic conditions, optimal temperature of 37^oC, pH value of 6-8, and genetic modification in the has enzymes to encounter the toxicity produced by the bacteria. The perspective sugar molecules for bacteria growth include glucose.

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